A basic technology in the field of molecular biology is the conversion of poly(A)+RNA (mRNA) to double-stranded (ds) complementary DNA (cDNA), which then can be inserted into a cloning vector for generating a cDNA library or expression in an appropriate host cell. Advances in cDNA library construction technology have made possible the discovery and production of a wide range of biologically important proteins.
Several procedures for generating cDNA libraries which have been used during the last 15 years are comprehensively reviewed in Wu, ed. Methods in Enzymology (1987), vol. 152. For the most part, cDNA library construction technologies use poly(A)+RNA as a starting material. The intact poly(A)+RNA is characterized by a polyadenylated "tail" at its 3' end and a characteristic "CAP structure or cap site" at the 5' end. A critical requirement for cDNA library construction is to completely copy poly(A)+RNA to full-length cDNA and retain the complete sequence information on the structure of the protein encoded by mRNA.
One generalized and commonly used method by which the poly(A)+RNA is copied into cDNA employs reverse transcriptase, which starts at the 3' end of the mRNA from an oligo d(T) primer and proceeds towards the 5' end to generate a cDNA:mRNA hybrid (Gubler et al., 1983). The RNA strand is then removed from the hybrid by action of RNase H and a second DNA strand is then synthesized using DNA polymerase I. The resulting heterogeneous mixture of ds cDNA molecules can then be cloned into suitable recombinant DNA vector molecules using a variety of techniques. Unfortunately, for the majority of mRNAs, this method does not allow for the synthesis of "full-length" cDNA because reverse transcriptase can not efficiently copy them into full-length cDNAs. The efficiency of copying is inversely proportional to the length of mRNA; thus, the problem of "full-length" cDNA synthesis is more acute for longer mRNAs. Moreover, the current technology can generate deletions at the 5' and 3' ends of the cDNA.
In an alternative approach, poly(A) tails of mRNA molecules are first annealed to oligo (dT) linking with linearized vector DNA (vector primer)(Okayama et al., 1982; Pruitt, International Patent, Appl. No. 89110816.9). Then, the first strand of cDNA synthesized by reverse transcriptase is tailed at the 3' end by oligo dt which facilitates subsequent cloning by circularization into vector primer. This method also generates greater numbers of cDNA clones that contain truncated cDNAs due to non-full-length cDNA synthesis.
As a result of the shortcomings using present technologies to generate conventional cDNA libraries, the majority of the cDNA clones lack sequences close to the 5' end of the mRNAs. This results in a loss of important information required to make functional proteins. Two selection procedures have been developed in efforts to enrich cDNA libraries for "full-length" cDNA clones. In CAP retention procedure (CAPture) cap-binding protein (eukaryotic initiation factor 4E) in combination with RNase A was used to purify full-length cDNA:mRNA hybrids (Edery et al., 1995; Sonenberg et al., U.S. Pat. No. 5,219,989). Shorter duplexes corresponding to non-full-length cDNA fragments are not selected, since the CAP structure of mRNA is removed from the RNA moiety by nuclease treatment. Although the CAPture method could potentially enrich cDNA libraries for clones containing the authentic 5' ends, the yield of full-length cDNA is very low, especially for long cDNAs (1-5%). The low yield is a significant disadvantage for this technology.
In the "oligo-capping" method, the CAP structure of mRNA is selectively replaced with an oligoribonucleotide, thus generating chimeric oligonucleotide/full-length mRNA intermediates which are subsequently used for synthesis of full-length cDNAs (Maruyama et al., 1994; Fromomt-Racine et al., 1993; Kato et al., International Patent, Publ. No. 0 625 572 A1, Appl. No. 93921061.3). However, this method is complicated, involving treatment of mRNA with an alkaline phosphatase, decapping mRNA with tobacco acid pyrophosphatase, and ligation of the oligonucleotide to the 5' end of mRNA by T4 RNA ligase. These multiple enzymatic steps degrade mRNA, thereby generating incomplete cDNA fragments for subsequent cloning procedures. Size distribution of cDNA inserts in cDNA libraries generated using the "oligo-capping" method is typically less than 3 kb. This is much less than full-length mRNA size distribution (Kato et al., 1994) and indicates the low efficiency of "full-length" cDNA cloning by "oligo-capping" technology.
As can be understood from the foregoing, conventional methods for constructing cDNA libraries containing full-length cDNA clones are restricted by low efficiency and the use of multiple, time-consuming steps. Accordingly, a simple method that would generate high quality, full-length cDNA from RNA is highly desirable.